Complementation. Scotland Received for publication 26 November been described previously (1, 2). Other strains are

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1 JOURNAL OF BACTERIOLOGY, June 1972, p American Society for Microbiology Vol. 110, No. 3 Printed in U.SA. Genetic Analysis of Transfer by the Escherichia coli Sex Factor F, Using P1 Transductional Complementation NEIL WILLETTS AND MARK ACHTMAN1 MRC Molecular Genetics Unit, Department of Molecular Biology, University of Edinburgh, Edinburgh, Scotland Received for publication 26 November 1971 P1 transduction has been used to perform a complementation analysis of a series of transfer-deficient mutants of Flac. The results define ten cistrons and are consistent with the results of a conjugational analysis presented in an accompanying report. Both sets of results are summarized here. Between them, they define eleven cistrons, traa through trak, necessary for conjugational deoxyribonucleic acid (DNA) transfer. Mutants in trai and trad and some in trag still make F-pili, although trad mutants are resistant to f2 phage; their products may be involved in conjugational DNA metabolism. Other mutants in trag and all mutants in the remaining eight cistrons do not make F-pili. One of these, traj, may be a control cistron, and the others may specify a biosynthetic pathway responsible for synthesis and modification of the F-pilin subunit protein and its assembly into the F-pilus. A previous paper (1) has described the isolation and characterization of a series of transfer-deficient mutants of Flac, and the accompanying paper (2) describes a complementation analysis using conjugation to construct F/F heterozygotes. This paper describes a similar analysis using P1 transduction to form transient heterozygotes. For this, strains carrying Tra- Flac mutants were transduced with P1 grown on strains carrying other Tra- Flac mutants; the resultant transductional F/F heterozygotes were immediately tested for their ability to transfer Flac to a Lac- P1R recipient strain. A positive result indicated complementation. The results obtained both agree with and supplement the results given in the accompanying report. They confirm the existence of eight of the nine cistrons defined there, and define two new cistrons, making a total of eleven. A summary of the cistron allocations by both techniques is given, and the possible functions of the various gene products are discussed. MATERIALS AND METHODS Bacterial strains. Derivatives of JC3272 carrying the mutant Flac episomes were used; these have been described previously (1, 2). Other strains are described in Table 1. Media. Most of the media used have been described previously (6). P1 was grown and assayed on LCTG agar plates using LC top agar (15). Spectinomycin sulfate was generously provided by G. B. Whitfield, The Upjohn Co., Kalamazoo, Mich. P1 techniques. P1 vir a (here called P1; see reference 15) was used throughout. Lysates were prepared by a confluent plate lysis technique, using an inoculum of 106 P1 JC3272 (i.e. P1 grown on the F- parental strain) and 0.5 ml of an overnight broth culture of the strain to be used as transductional donor. These lysates generally contained 1011 to plaque-forming units (PFU)/ml; all lysates were reassayed the day before being used in a complementation experiment. P1 was assayed by mixing 0.1 ml of appropriate dilutions with 0.1 ml of an overnight culture of an Escherichia coli K-12 strain grown in L broth containing M CaCl2 and 0.01 M MgSO4. A 1.5-ml amount of LC top agar was added, and the mixture was poured onto an LCTG plate. This technique was simple and gave optimal plating efficiency. Transductional complementation tests. An exponentially growing culture of a mutant Flac derivative of JC3272 at about 1.2 x 108 cells/ml was centrifuged and suspended in one-fifth volume of 0.5% tryptone containing M MgSO4 and M CaCl2. A 0.2-ml amount of this was mixed in a test tube (18 by 150 mm) with 0.1 ml of a P1 lysate diluted to 1.2 x 109 PFU/ml in L broth. After 15 min 'Present address: Max-Planck Institut fbr Biologie, 74 of incubation, 1.7 ml of an exponentially growing Tilbingen, Corrensstrasse 38, West Germany. culture of JC5465 (SpcRLac- P1RF-), at about 3 x 843

2 844 WILLETTS AND ACHTMAN J. BACTERIOL. TABLE 1. Bacterial strainsa Strain Episome Lac Gal His Trp Spc Str Other JC3272 F- S R Lys- JC3273 Flac +/ S R Lys- JC5462 F R R PJR JC5465 F R S PJR JC5771 Fdjac S S ED12 F /- - S R Lys- ED14 F8 - +/- - - S R Lys- ED17 F S R Lys- ED80 Ft.,ac +/ S S AB259 (Hfr H) S S P4x (Hfr P4x) S S Met- (+) athe nomenclature used is described by Achtman et al. (2). JC3273, ED12, ED14, and ED17 are derivatives of JC3272. JC5465 is a spontaneous PlR mutant of JC5455 (2), and JC5462 is a PlR Trp+ derivative of JC3054 (2). ED80 (Ft..11ac/PS200) was obtained from Y. Hirota, and JC5771 (Fdiac/PS200) from F. Cuzin, to whom we are grateful. 108 cells/ml, was added. This gave a donor-recipient ratio of approximately 1:4. After 60 min of incubation to allow Flac transfer, 0.5-ml portions (or 0.1 ml for trad mutants) were plated by using the soft agar overlay technique on medium selective for Lac + [SpcRJ progeny. The plates were incubated for 2 days before counting, and usually 20 to 400 colonies per plate were obtained. The experiments were performed at 42 C throughout except that the plates were incubated at 37 C. RESULTS Optimal conditions for complementation analysis. Three factors seemed likely to affect the efficiency of transductional complementation and its detection: the multiplicity of P1 infection, the length of the transduction period, and the length of the mating period with the recipient strain, JC5465. The optimal values for these parameters were determined in a series of experiments using P1 grown on JC3273 (which carries wild-type Flac) to transduce three Tra- Flac mutants representative of the three phenotypic groups defined by the pattern of sensitivity to male-specific phages (1). Complementation was then assayed by mating the transduced cultures with JC5465 and selecting Lac+ [SpcR] progeny. In the first series of experiments, P1 *JC3273 was added at a multiplicity of one to cultures of the three strains. After incubation for various times, an exponential culture of JC5465 was added and a period of 60 min was allowed for Flac transfer. In all cases, a 15-min transduction period was long enough for subsequent efficient Flac transfer (Fig. la). In a second series of experiments, a 15-min transduction period was followed by varying periods of mating. Sixty minutes was long enough to give near-maximum results (Fig. lb). Finally, using these conditions, the optimal multiplicity of P1 infection was determined (Fig. 2). As the multiplicity was increased from 102 to 1 there was an almost commensurate increase in the number of Lac+ [SpcR] progeny per portion plated, the number of progeny per 106 P1 decreasing only slightly. At higher multiplicities there was no further increase in the number of progeny per portion plated, corresponding to a steep fall in the number of progeny per 106 P1. A 15-min transduction period using a P1 multiplicity of one, followed by a 60-min mating period, was therefore used in all the complementation experiments described below. In all cases, a correction was made for the residual transfer level of the recipient mutant used, by performing a parallel experiment with P1 grown on JC3272 (the F - parental strain) and subtracting the number of Lac+ [SpcR] progeny found from the corresponding experimental values. Furthermore, day-to-day variations were minimized by expressing the corrected results as a percentage of that obtained by transducing the same recipient culture with P1 grown on JC3273 (which carries wild-type Flac). Complementation tests with reference strains. Preliminary tests using a large number of mutants allowed most to be tentatively assigned to ten complementation groups or cistrons. These include two new cistrons, designated trai and trak, which were not defined in the conjugational complementation analysis (2) since no suppressible mutations in these genes have been found. Only one trak mutant, JCFL105, was available, and only one trai mutant, JCFL65, had a residual transfer frequency low enough to allow its use as recipient in transductional complementation anal-

3 VOL. 110, 1972 TRANSFER: TRANSDUCTIONAL COMPLEMENTATION ANALYSIS 1OOC 6) (b) 845.,,l,It 5 <X>==S X.1E Time o fraosuction (mn) Time of mc*ing (min) FIG. 1. Optimum parameters for complementation. Complementation experiments using different periods of transduction (a) and mating (b) were performed as described in the text for the JC3272 derivatives of JCFL25 (0), JCFL8 (0), and JCFL42 (A), belonging to phenotypic groups I, II, and III, respectively. 10 w taaiplicity of Pi Wection FIG. 2. Variation of multiplicity of infection. Complementation experiments using different multiplicities of Pl infection were performed as described in the text for the JC3272 derivatives of JCFL25 (0), JCFL8 (0), and JCFL42 (A), belonging to phenotypic groups I, II, and III, respectively. ysis. The cistron traj, defined by conjugational complementation (2), could not be clearly defined here since the only traj mutant with a low enough residual level of transfer was too poorly complemented to use as transductional recipient. With these exceptions, two reference strains representing each group were chosen on the basis of their low levels of residual transfer and their efficient complementation with P1l JC3273. Reciprocal complementation experiments were then completed between these reference strains using the conditions described above. The results are shown in Table 2. If the mutations carried by the Flac mutants in the donor and recipient strains were identical or in the same cistron, little or no complementation was observed. However, if the mutations belonged to different cistrons, elevated numbers of Lac+ [SpcR] progeny were usually found, indicating complementation. In a very few cases, for example, trae7-traa25, a cross gave a low or negative result, whereas its reciprocal gave a positive result; such a situation was interpreted as demonstrating complementation. With these exceptions, all results were consistent and confirmed the assignment of the mutations to ten complementation groups. The actual levels of complementation observed, although repeatable, varied widely depending upon the cistrons or, in some cases,

4 846 WILLETTS AND ACHTMAN J. BACTERIOL. TABLE 2. Reciprocal complementation tests between reference mutationsa Donor tra Recipient tra mutation mutation Al A25 B2 B62 C6 C33 D8 D38 E7 E18 F13 F114 G42 G79 H55 H88 I65 K105 A A B B C C D D E E F F G: G H H I I J J K ajc3272 derivatives carrying the mutant Flac episomes were used, and their strain numbers are given in references 1 and 2. The results were corrected by subtracting the values obtained in parallel experiments using P1-JC3272 and are expressed as percentage of the correspondingly corrected values found using P1- JC3273. All results are the average of two separate experiments. the mutations, involved, and their significance cannot at present be evaluated. The results for two mutants in traj, used as transductional donors, are included in Table 2. As expected, they complemented all mutants in the ten complementation groups described above. Efficiency of complementation. Approximately 4 x 10-6 Lac+ [SpCR] progeny were obtained per P1 JC3273 for all transfer mutations except those in trad (Table 3). For these, approximately tenfold higher levels were always found. The reason for this difference is not clear, especially since it was not observed in conjugational complementation experiments (2) Ṫhe number of progeny must depend upon the frequency of transduction of the wild-type allele, the efficiency of complementation, and the efficiency of transfer of Flac. If the assumption is made that the frequency of transduction of an episomal tra+ allele is similar to that found for chromosomal markers, that is 10-5 to 10-6 per P1, then, since the efficiency of transfer of Flac under the conditions used was about 100%, the efficiency of complementation must also be high, probably near 100%. Most of the Flac transfer in these complementation tests was indeed due to complementation rather than to recombination. To show this, 50 Lac+ [SpCR] clones from experiments in which P1 JC3273 was used to transduce each of the reference strains were patched on the same selective medium. After overnight growth, the plates were replicated onto L plates, and these were incubated for 6 to 8 hr. The transfer ability of the clones was then tested in a replica-plate mating with JC5462 (F- Lac- His- PJR SpCR StrR), selecting Lac+ [Trp+Stra] progeny (Table 3). Most of the Lac+ [SpcR] clones were still transfer-deficient, indicating that complementation had been responsible for their formation. A number of clones carrying Tra+ recombinants were seen,

5 VOL. 110, 1972 TRANSFER: TRANSDUCTIONAL COMPLEMENTATION ANALYSIS TABLE 3. Efficiency of complementationa tra Mutation in Lac+ [SpCR'] tra+ Recombirecipient strain progeny per Lac+ [SPCRl clones traal traa trab trab trac trac trad trad trae trae traf traf trag trag trah trah trai trak ajc3272 derivatives carrying the Flac mutants (see references 1 and 2 for the strain numbers) were used in complementation experiments with P1- JC3273 as described in the text. The results have been corrected for residual transfer and reversion, and represent the average of 6 to 15 experiments in each case. however, at a frequency varying from <2 to 4%. Lac+ [SpcRI clones arising in the reciprocal complementation tests between the reference strains were also tested and gave similar results. When JC3272, carrying the lac deletion X74 (5), was infected with P1 * JC3273, no Lac+ transductants (< per P1) were obtained, although near-normal numbers were observed when it was infected with P1 grown on a lac+ F- strain or when a lac- point mutant was infected with P1.JC3273. Flac is therefore too large to be transduced as a whole, and the Lac+ transductants in the latter two cases presumably were chromosomal recombinants. The absence of Lac+ transductants in the first case also indicates that, although a chromosome carrying the X74 deletion is mobilized by Flac (5), the chromosomal segment carried by this episome does not completely span the X74 deletion. In the transductional complementation experiments, then, only the resident mutant Flac episome could be transferred from the F/F heterozygotes and give rise to Lac+ [SpcRJ progeny. In contrast, both resident and incoming Flac mutants could be transferred from the F/F heterozygotes formed by conjugation (2). Complementation tests with other Flac mutants. Many other mutants were tested for 847 complementation in reciprocal experiments with the reference strains. However, some of the mutants with high residual transfer levels could not be successfully used as recipients, since the number of progeny resulting from residual transfer (using P1 JC3272) was similar to the number obtained using P1 *JC3273. These mutants could still be used as transductional donors with the reference strains, since only the resident mutant Flac episome was transferred from transductional F/F heterozygotes. Similarly, poorly complemented mutants which sired very few Lac+ [SpcR] progeny after transduction with P1 *JC3273 could be used only as donors. Although a comprehensive list of such mutants is not available since not all were tested, it includes four of the five dominant mutants mentioned in the accompanying paper (2); the other mutant, JCFL34, was recessive in transductional tests. Several other mutants were poorly complemented in transductional complementation experiments but not in the corresponding conjugational experiments; this probably reflects the greater sensitivity of the latter method. The results obtained, together with those from conjugational complementation experiments, are summarized in Table 5. Most mutant episomes carried a single mutation in 1 of the 11 tra cistrons, and when a given mutant was tested by both conjugational and transductional techniques, the same cistron assignment was almost invariably made. A few of the nitrosoguanidine-induced mutants carried two mutations ifi two different cistrons, and one, JCFL72, carried mutations in three different cistrons. In two cases, the transductional analysis gave slightly different results from the conjugational analysis. JCFL32 appeared to carry a traa mutation, whereas in the conjugational analysis, it gave intermediate results when used as recipient in complementation tests with the amber mutation, traal. This discrepancy is probably due simply to the lower sensitivity of the transductional technique. JCFL57 appeared to carry mutations in trac and trae when used as transductional donor but only in trac when used as transductional recipient. It may carry a leaky trae mutation. This would also account for the low but significant level of complementation between JCFL57 and an amber trae mutant in conjugational complementation experiments and for its partial suppression in a SUUGA+ host (2). JCFL51 and JCFL72 carry dominant nonsuppressible mutations and were therefore not classifiable by conjugational complementation

6 848 WILLETTS AND ACHTMAN J. BACTERIOL. analysis. They could, however, be used as transductional donors; JCFL51 carried a mutation in trac, and JCFL72 carried three mutations, in traa, trac, and trah. Multiply deficient nonsense mutants. JCFL4, carrying the recessive amber-suppressible mutation tra-4, seemed to lack the products of six cistrons, trab, trac, traf, trag, trah, and trak, as well as having lost surface exclusion (1). This multiple loss, also found in the conjugational analysis (2), may be due to polarity in a polycistronic messenger ribonucleic acid (RNA) coding for the products of these cistrons. This hypothesis was strengthened by the finding that the six cistrons map together (7a), as does the surface exclusion gene (N. Willetts, unpublished data). trak is probably the first cistron in the proposed operon to be transcribed (7a), and tra-4 may be an extreme polar trak mutation. When used as transductional donors, JCFL28 and JCFL29, carrying the nonsense mutations tra-28 and tra-29, gave no complementation with JCFL4 and seemed to lack the same six tra cistron products. They also showed lowered levels of surface exclusion (see footnote b to Table 5). When used as recipients in transductional complementation tests, however, they gave positive results with all tra mutants except JCFL4 and those carrying mutations in trab and trak. Similarly, when used as recipients in the more sensitive conjugational complementation tests, they showed no complementation with JCFL4 and intermediate levels of complementation with trab mutants; trak mutants could not be tested, and all other mutants gave positive results (2). It is possible that tra-28 and tra-29 are also polar trak mutations which are quantitatively not as strongly polar as tra-4. The F-like plasmid R100-1 could provide the missing functions for each of JCFL4, JCFL28, and JCFL29, for both Flac transfer and F-pilus formation. This shows that none of the tra cistrons affected by the tra-4, tra-28, and tra-29 mutations are plasmid-specific and is consistent with these mutations being polar trak mutations, since neither the product of trak (Willetts, unpublished data) nor the products of the other five tra cistrons affected (13) are plasmid-specific. Complementation by other male strains. P1 lysates were made on JC3272 derivatives carrying F (ED17), the Fhis element F57 (ED12) or F8 (ED14), as well as on two Hfr strains, Hfr H (AB259) and P4x. These lysates were used in complementation experiments with all the reference strains. Representative results are shown in Table 4. Normal complementation was observed with F8, F57, and both Hfr strains, although Hfr H gave rather high levels. The F+ derivative gave unexpectedly poor complementation in view of its high transfer ability measured in conjugation experiments (250% in 30 min) and good complementation (equivalent to F57) in conjugational complementation experiments. Poor transductional complementation is perhaps due to an abnormally low number of P1 transducing particles carrying F DNA, although this was not observed by Arber (3). Other transfer-deficient F mutants. One of the first transfer-deficient mutants of F to be described and Fd5lac (9). A strain carrying this mutant episome (JC5771) was used as donor in transductional complementation tests with the reference strains (Table 4); it was too poorly complemented to use as recipient. No complementation was observed, and Fd5lac may be mutant in a control element or may carry a polar or deletion mutation affecting all the tra cistrons tested. Another mutant of interest is F,,621ac, isolated as a temperature-sensitive replication mutant. It is also transfer-deficient, and this has been taken as an indication of a connection between replication and transfer (8). It carries a mutation in trag (Table 4), presumably separate from the replication mutation, since other trag mutants are replication-proficient at 42 C. This also follows from the temperature sensitivity of replication, but not of transfer, of Ft862lac (4, 14). Ohtsubo et al. (11) have used transfer-deficient mutants of both F8 (an Fgal) and R100-1, a related but compatible sex factor, to perform a genetic analysis of conjugation in stable heterozygotes. However, the assumption that one sex factor can supply the wild-type allele of a mutant allele in a different sex factor is not always justified (13, 14). For purposes of comparison, one transfer-deficient F8 mutant of each of the classes described by Ohtsubo et al. (11) has been used as transductional donor with the reference strains (Table 4). The six complementation groups called A, B, C, D, E, and F by them probably correspond to the cistrons we have designated trai, trad, trag, traf, trac, and trae, respectively. However, the F8 mutant in JE3445 which they assigned to group B carries mutations in both trai and trad, and the F8 mutant in JE3431 which they assigned to group D carries mutations in both traf and trak. In addition, Ohtsubo et al. (11) described two other groups of F8 mutants,

7 VOL. 110, 1972 TRANSFER: TRANSDUCTIONAL COMPLEMENTATION ANALYSIS P1 donor otrain I TABLE 4. Complementation by other male or transfer-deficient strains" tra Mutation in reference strain used as recipient Episomeb A25 B2 C6 D8 E7 F13 G42 H55 I65 K105 ED12 F ED14 F ED17 F < 1 AB259 (HfrH) P4x (Hfr P4x) JC5771 Fd5laC ED80 Ft,,*ac JE3513 F8-N33 (A) JE3445 F8-N73 (B) JE3453 F8-N31 (C) JE3431 F8-M9 (D) JE3517 F8-N37 (E) JE3274 F8-M1 (IF) JE4574 F8-N62 (G) JE3512 F8-N32 (H) a See footnote to Table 2. All negative results were confirmed using a second reference strain as recipient. The results with F8 mutants are expressed as a percentage of the value found using P1 -ED14 (carrying wildtype F8). bthe strains with JE numbers were obtained from E. Ohtsubo, to whom we are grateful, and carry transfer-deficient F8 mutants designated as listed. The letters in parentheses indicate the groups to which these mutants were assigned by Ohtsubo et al. (11). called G and H, for which corresponding R100- phenotypes of all the transfer-deficient Flac 1 mutants were not found. One G mutant, mutants tested. All mutants in eight cistrons JE4574, carried a mutation in trab, and an H and some in a ninth, trag, were completely mutant, JE3512, was not assignable to any of resistant to male-specific phage. This probably our cistrons. The results with JE3431, JE3274, results from inability to make the F-pilus, JE3453, and JE3512 were confirmed by transferring the mutant episomes carried by these electron microscope (C. Brinton, and M. Acht- since F-piliated cells could not be seen in the strains to JC3272 and using these derivatives man, unpublished data). Although the F-pilus as recipients in conjugational complementation tests. this carries both phosphate and glucose resi- is constructed from a single protein subunit, The correlations found between the transferdeficient F8 mutants and the Flac mutants are seems likely that one of the nine cistrons codes dues (Brinton, personal communication). It consistent with the phenotypes of the strains for the F-pilus subunit protein, and others for and the mapping data (7a, 10). its phosphorylation and glucosylation. Still others may be involved in the formation of any DISCUSSION The transductional technique for performing the complementation analysis has the advantage, compared to the conjugational method, that reciprocal crosses involving nonsuppressible mutations are possible. In particular, mutants belonging to complementation groups for which an amber mutant was not available (tral, trak) could be unequivocally classified. It is, however, limited in its sensitivity, due to the low frequency of transduction, so that leaky mutants or poorly complemented mutants could not be used as recipients. However, even these types of mutants could be used as transductional donors. Table 5 gives the cistron assignments and 849 intracellular structure necessary for construction of the F-pilus. Mutations in one of the nine cistrons, traj, are pleiotropic; the traj product is required not only for F DNA transfer and F-pilus formation but also for surface exclusion (2). At a genetic level, it may be required for transcription or translation of traa, trai, the surface exclusion gene and perhaps other tra cistrons, or it may form a multienzyme complex with their products which is necessary for expression of their activities (13). The products of three other cistrons, trad, trag (in part; see reference 2) and trai, are not required for F-pilus formation. They may determine steps in DNA metabolism such as nicking the covalently closed Flac DNA circles normally found in the cell (7) to allow linear single-stranded DNA transfer and the replica-

8 850 WILLETTS AND ACHTMAN J. BACTERIOL. TABLE 5. Cistron Male-specific Surface Cistron assignments Episome JCFL numbersc phage phenotypea exclusione C traa I + l(a)d e, 25(A)e, 44, 107(F). trab I + 2(A)d,e, 16(A)d, 45f, 62e, 68, 69, 71f, 73, 78(A)e, 108(F), 109(F), 113(F). trac I + 3(A)d e, 5(A)d. e 6(A)d. e, 12(A)d, 15(A)d, 17(A)d, 20(A) d, 30e, 33e, 34e 46f' 48e, 49e, 50f 51', 52f' 70e, 74(U), 77, 99, 117(F). trad I + 8(A)d e, 9, 14(A)d, 36e, 37( )e, 38e, 39f, 9, 47e, 58'f 60e, 63, 64, 83(A), 89(A), 115(F), 116(F). trae I + 7(A)e, 18(A)d e, 59', 67f 111(F)9 112(F). traf I + 13(A)d e943(a)f, 110(F), 114(F)e. trag I + 42e,79(A)d, e h, 98,100(F), lol(f). trag I + 24(A)d., 81(A)d {, 86(A)d.f, 104(F), 106F). trah I + 27e, 54e, 55e 66t 76', 80(A)d, 82(A)d, 88(A)d e, 96. trno m + 40', 41t, 65e,102(F)'. traj I - 26f, 10, 90(A)d,. trnk I + 105(F) e. Multiple' I 56t, 3l, 35e9 57e9 103e, 75', 53' 72'. Other I 4(A) d. e 28(U) e, 29(O) e 32e. athe male-specific phage phenotypes are: I, flr f2r Q#R; II, fls f2r Q's; IH, fls f2sqfs. The plus or minus for surface exclusion is deduced from the results previously presented in Table 2 of reference 1. Further relevant surface exclusion indexes not quoted there are: JCFL45 (830), JCFL81 (300), JCFL105 (800), JCFL75 (2.6), JCFL28 (25), JCFL29 (20), and JCFL32 (170). c The tra mutation numbers are not given, but are the same (by design, but not of necessity) as the JCFL numbers, except for the multiple mutants (see footnote i). A letter in parentheses indicates a suppressible or frameshift tra mutation: A = amber, 0 = ochre, U = UGA, F = frameshift (i.e., induced by ICR 191). In the complementation analysis, mutants were tested against representatives of all 11 complementation groups, except trai and trak when used as recipients in conjugational complementation experiments, and traj when used as donors in transductional complementation experiments. Also, only the reference mutants listed in Table 2, multiple mutants, and undesignated mutants were tested in transductional complementation experiments with trak. All mutants were used as recipients in conjugational complementation analysis (except JCFL51 and JCFL72). d Indicates that the mutant was also used as donor in conjugational complementation experiments. eindicates that the mutant was also used as both donor and recipient in transductional complementation experiments. ' Indicates that the mutant was also used as donor in transductional complementation experiments. 'The mutation trad39 carried by JCFL39 makes transfer temperature-sensitive (1). h The mutation trag79 carried by JCFL79 gives only partial male-specific phage sensitivity (1). IThe tra mutations carried by the multiple mutants are: JCFL56 (A56, C203); JCFL31 (B31, C207); JCFL35 (B35, H206); JCFL57 (C57, E205); JCFL103 (C103, E208); JCFL75 (C75, J204); JCFL53 (E53, H202); and JCFL72 (A72, C209, H210). tion of F DNA in the donor cell which is found associated with conjugation (12). The multiple loss of the products of trab, trac, traf, trag, trah, and trak and the surface exclusion gene produced by the amber mutation tra-4, and partially by the nonsense mutations tra-28 and tra-29, suggests that these seven cistrons form a single operon. All other presumptive polarity effects observed so far involved these same seven cistrons (2). Further investigations of the operon structure of the 11 tra cistrons and the control of their expression are in progress. ACKNOWLEDGMENTS We are most grateful to Sheelagh McQuitty for her expert technical assistance. One of us (M.A.) was supported by a Canadian Medical Research Council Fellowship. LITERATURE CITED 1. Achtman, M., N. S. Willetts, and A. J. Clark Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherichia coli by isolation and characterization of transfer-deficient mutants. J. Bacteriol. 106: Achtman, M., N. Willetts, and A. J. Clark Conjugational complementation analysis of transferdeficient mutants of Flac in Escherichia coli. J. Bacteriol. 110: Arber, W Transduction of chromosomal genes and episomes in E. coli. Virology 11: Cuzin, F., G. Buttin, and F. Jacob On the mechanism of genetic transfer during conjugation of E. coli. J. Cell Physiol. Supp. 1 70: Cuzin, F., and F. Jacob Deletions chromoso-

9 VOL. 110, 1972 TRANSFER: TRANSDUCTIONAL COMPLEMENTATION ANALYSIS miques et integration d'un episome sexuel Flac chez E. coli K12. C. R. Acad. Sci. Paris 258: Finnegan, D. J., and N. S. Willetts Two classes of Flac mutants insensitive to transfer inhibition by an F-like R factor. Mol. Gen. Genet. 111: Freifelder, D Studies on E. coli sex factors. HI. Covalently closed Flac DNA molecules. J. Mol. Biol. 34: a. Ippen-Ihler, K., M..Achtman, and N. Willetts Deletion map of the Escherichia coli K-12 sex factor F: the order of eleven transfer cistrons. J. Bacteriol. 110: Jacob, F., S. Brenner, and F. Cuzin On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28: Jacob, F., and E. L. Wollman Sexuality and the genetics of bacteria. Academic Press Inc., New York. p Ohtsubo, E Transfer-defective mutants of sex factors in E. coli. II. Deletion mutants of an F prime and 851 deletion mapping of cistrons involved in genetic transfer. Genetics 64: Ohtsubo, E., Y. Nishimura, and Y. Hirota Transfer-defective mutants of sex-factors in E. coli. I. Defective mutants and complementation analysis. Genetics 64: Vapnek, D., and W. D. Rupp Asymmetric segregation of the complementary sex-factor DNA strands during conjugation in E. coli. J. Mol. Biol. 53: Willetts, N. S Plasmid specificity of two proteins required for conjugation in E. coli K12. Nature N. Biol. 230: Willetts, N. S., and P. Broda The E. coli sex factor, p In Wolstenholme and O'Connor (ed.), Ciba foundation symposium on bacterial episomes and plasmids. Churchill, London. 15. Wolf, B., A. Newman, and D. Glaser On the origin and direction of replication of the E. coli K12 chromosome. J. Mol. Biol. 32: Downloaded from on November 16, 2018 by guest